Electrochemical sensor based on PEDOT/CNTs-graphene oxide for simultaneous determination of hazardous hydroquinone, catechol, and nitrite in real water samples

Hydroquinone (HQ), catechol (CC) and nitrite (NT) are considered aquatic environmental pollutants. They are highly toxic, harm humans’ health, and damage the environment. Thus, in the present work we introduce a simple and efficient electrochemical sensor for determination of HQ, CC, and NT simultaneously in wastewater sample. The sensor is fabricated by modifying the surface of a glassy carbon electrode (GCE) by two successive thin films from poly(3,4-ethylenedioxythiophene) (PEDOT) and a mixture of carbon nanotubes-graphene oxide (CNT-GRO). Under optimized conditions the HQ, CC, and NT are successfully detected simultaneously in wastewater sample with changing their concentrations in the ranges (0.04 → 100 µM), (0.01 → 100 µM) and (0.05 → 120 µM), the detection limits are 8.5 nM, 3.8 nM and 6.1 nM, respectively. Good potential peak separations: 117 mV and 585 mV are obtained between the HQ-CC, and CC-NT. The sensor has an excellent catalytic capability toward the oxidation of HQ, CC, and NT due to good synergism between its composite components: PEDOT, GRO and CNTs. The features of the sensor are large active surface area, good electrical conductivity, perfect storage stability, good reproducibility, anti-interference capability and accepted recovery rate for HQ, CC, and NT determination in wastewater sample.


Preparation of GRO
Hummer's method is used to prepare GRO.Briefly, to prepare the pre-oxidized graphite (98%), H 2 SO 4 (40 mL), K 2 S 2 O 8 (8.4 g) and P 2 O 5 (8.4 g) are used to cure 10 g of highly pure graphite.Then in an ice bath 3 g of dried preoxidized graphite is stirred with 115 mL of concentrated H 2 SO 4 for 10 min.This is followed by gradual addition of 15 g of KMnO 4 while stirring for a period of two hours.After dilution with water and treatment with H 2 O 2 , a bright yellow color suspension is obtained and filtered.Lastly, 1:10 (v/v) HCl-solution is used to wash the suspension, and is dried in an oven overnight at 80 °C42 . www.nature.com/scientificreports/

Preparation of the sensor
The GC/PEDOT/CNT-GRO sensor is prepared as follows: (GC/PEDOT) is prepared from a solution of 0.001 M EDOT and 0.05 M Bu 4 NPF 6 in acetonitrile by electrochemical polymerization 43,44 .This step is accomplished by using cycling voltammetry (CV) in the potential range (− 1.5 V to 2 V) for 3 cycles at scan rate of 100 mV s −145 .Then 10 μL from a suspension mixture of (0.5 mg GRO-0.5 mg CNTs/1.0 mL DMF) is drop-cast over the previous modified electrode surface GC/PEDOT.Then the electrode is dried in an oven at 50 °C for 10 min.
We run CV experiments in 1.0 mM K 3 [Fe(CN) 6 ] system using the modified electrodes to calculate their electrochemical effective surface areas as mentioned elsewhere 46 .The values of the active surface areas for bare GC, GC/GRO, GC/PEDOT, GC/PEDOT/GRO, GC/CNT, GC/CNT-GRO, GC/PEDOT/CNT, and GC/PEDOT/ CNT-GRO are: 0.0474 cm 2 , 0.064 cm 2 , 0.0902 cm 2 , 0.0961 cm 2 , 0.102 cm 2 , 0.129 cm 2 , 0.1335 cm 2 , and 0.1462 cm 2 , respectively, Supplement Fig. 1A-H.The active surface areas of the modified electrodes increased in the following order GC < GC/GRO < GC/PEDOT < GC/PEDOT/GRO < GC/CNT < GC/CNT-GRO < GC/PEDOT/CNT < GC/ PEDOT/CNT-GRO.Modification of GC electrode surface increases the composite surface area, the contact area with analytes and improves the electrocatalytic behavior of the composite toward oxidation of HQ, CC, and NT.Thus, the oxidation current response of the analyte under investigation increases with increasing the electroactive surface area.

Preparation of real sample
We obtained water samples from river, the sample is pretreated as mentioned elsewhere 47 .The wastewater sample is diluted by 0.1 M PBS/pH 7.0 one time; 10.0 mL of diluted sample is transferred to the electrolytic cell.To reach the desired concentrations, the prepared solution 0.1 mM for each of HQ, CC, and NT mixture/0.1 M PBS/pH 7.0 is spiked with pre-calculated volumes in diluted wastewater sample.Then, the experiments are studied using DPV technique.

SEM and Raman analyses
Scanning electron microscopy (SEM) is used to investigate the surface structural/morphological images of the studied composites.We prepared thin films from GC/PEDOT, GC/PEDOT/GRO and GC/PEDOT/CNT-GRO over GC sheets as mentioned earlier, the films were introduced directly to the measurements without further treatment.
Surface enhanced Raman spectroscopy (SERS) is used to obtain the vibrational modes of the functional groups of the composite film.We prepared the composite sample GC/PEDOT/CNT-GRO over a GC sheet as mentioned earlier, and then the experiment is conducted on the prepared surface to record the Raman spectrum.

Surface characterization of the modified electrodes
The surface morphology of GC/PEDOT, GC/PEDOT/GRO and GC/PEDOT/CNT-GRO are examined by scanning electron microscopy.The morphology of PEDOT modified glassy carbon electrode surface shows a dense compact and homogeneous structure 48,49 , Fig. 1A.GRO sheets are characterized by thick flat flake layers, irregular shapes and rough surfaces as shown in Fig. 1B.The unwrinkled and disordered structures of GRO sheets are due to the presence of functional groups containing oxygen atoms over their surfaces.Figure 1C shows the SEM of GC/PEDOT/CNT-GRO, where the CNTs are thoroughly mixed with the graphene oxide sheets.Due to noncovalent π-π stacking interactions, the GRO sheets are surrounded by the CNTs tubular networks 50 .The presence of all elements in the composite are identified from EDX and mapping of the GC/PEDOT/CNT-GRO as shown in Fig. 1D-G.
The FTIR technique is used to analyze the structural components of PEDOT, CNT-GRO, and PEDOT/CNT-GRO.Supplement Fig. 2A shows the FTIR spectrum of PEDOT bands.Characteristic peaks are observed below 1600 cm −1 .The peaks at 1584 cm −1 and 1366 cm −1 are assigned to C=C and C-C stretching vibrations, while the bands at 1183 cm −1 , 1135 cm −1 , 1100 cm −1 , and 1056 cm −1 are attributed to the C-O-C bending vibrations in the ethylenedioxy group.The bands at 933 ~ 678 cm −1 are characteristic for stretching vibrations of the C-S-C bond in the thiophene ring, indicating a successful formation of PEDOT in the polymerization reaction 51,52 .Supplement Fig. 2B shows the FTIR spectrum of CNT-GRO composite.The spectrum consists of vibrational groups of the GRO that includes carbonyl (C=O), aromatic (C=C), carboxyl (COOH), epoxy (C-O-C), and hydroxyl (O-H) groups.A sharp peak appeared at 3420 cm −1 corresponds to the hydroxyl group (O-H) 53 .The peaks at 1697 cm −1 and at 1300 cm −1 are attributed to the ketone group (C=O) and to C-O-C 53 respectively.Additionally, the peak at 1577 cm −1 is attributed to the characteristic backbone C=C skeletal stretching of CNTs 54 .Supplement Fig. 2C shows the FTIR spectrum of PEDOT/CNT-GRO.The characteristic vibrational peaks that appeared are for PEDOT, CNTs, and GRO such as C-S-C at 887 ~ 684 cm −1 , C-C at 1381 cm −1 , C-O-C at 1184-1048 cm −1 , C=C at 1579 cm −1 , OH at 3419 cm −1 , and C=O at 1629 cm -1 , these peaks are slightly shifted compared to PEDOT and CNT-GRO spectra due to interactions between the composite components.
The Raman fingerprints of PEDOT, CNTs and GRO have been previously investigated [55][56][57] .The vibrational modes of PEDOT are located at 1514 cm −1 , 1390 cm −1 , and 1272 cm −1 , and assigned to the Cα = Cβ asymmetrical, Cα = Cβ symmetrical stretching, and Cα -Cα inter-ring stretching vibrations, respectively, Supplement 3A.The Raman spectrum of the CNTs shows the D-band peak between 1300 cm −1 and 1400 cm −1 spectral range, also known as the disorder band, is due to scattering from sp2 carbon containing defects precisely appeared at 1359 cm −1 .The peak between 1500 cm −1 and 1600 cm −1 region called the G-band appeared at 1573 cm −1 .A characteristic peak at 2688 cm −1 is also obtained and commonly written as D′ or 2D band, which depends upon the strain or stress applied to the carbon nanotube, Supplement 3B.The Raman spectrum of GRO displayed two unambiguous bands at 1348 cm −1 and 1585 cm −1 that are due to the D and G peaks, these bands arose from the Vol:.(1234567890 www.nature.com/scientificreports/lattice defects in the atomic crystal structure of the carbon material, Supplement 3C.To investigate the carbon structure and to confirm the presence of all the individual components in the composite, Raman spectrum of PEDOT/CNT-GRO is recorded (Fig. 1H).The characteristic peaks of the PEDOT are observed at 1381 cm −1 and 1514 cm −1 for the asymmetric and symmetric C=C stretching vibrations 58 .A characteristic peak of CNTs at 2688 cm −1 is obtained and commonly is written as D' or 2D band, which depends upon the strain or stress applied to the carbon nanotubes 59,60 .The GRO nanosheets exhibit the characteristic peaks at 1381 cm −1 (D band) for in-plane bond stretching of sp 2 carbon atoms and at 1568 cm −1 (G band) for defects of structure and lattice www.nature.com/scientificreports/distortion 61 .Supplement Fig. 4A displays the XRD pattern of GRO prepared by Hummer's method, where the GRO shows a distinctive diffraction peak at 10° corresponding to the (001) plane.Supplement Fig. 4B displays the XRD pattern of PEDOT/CNT-GRO where a major diffraction peak appeared at 2θ = 26°, that is attributed to the (002) plane.The sharpness of this peak is indicative of the structure of multiwalled carbon nanotubes 62 .Also, there is another peak for CNTs which appeared at 42.7°, that is corresponding to (100) plane.PEDOT should show a relatively medium peak at around 2θ = 26°, which is overlapping in this case with the sharp peak of carbon nanotubes 63 .Besides, the pattern shows a diffraction peak appeared at 2θ = 10° for GRO which is assigned to the (001) plane 64 .

Electrochemical impedance spectroscopy "EIS"
Electrochemical impedance spectroscopy is an effective technique to investigate the interface properties of the modified electrodes.A suggested illustration of the surface modification and its corresponding variation in the charge transfer kinetics and capacitive components of the system can be clarified by the aid of EIS.EIS experiments are carried out in 1.0 mM K 3 [Fe(CN) 6 ]/0.1 M PBS/pH 7.0 at applied potential of 0.1 V (vs.Ag/AgCl) in the frequency range of 0.1 Hz to 100 kHz at bare GC, GC/PEDOT, GC/CNT, GC/CNT-GRO, GC/PEDOT/ CNT, and GC/PEDOT/CNT-GRO electrodes.Figure 2 shows the impedance spectra in the form of Nyquist plots for the different working electrodes.The software used for EIS data fitting is supplied with the instrument.Supplement Fig. 5 shows three equivalent circuits used for fitting the EIS experimental data.The first equivalent circuit is used for fitting the GC electrode data, the second equivalent circuit is used for fitting the GC/PEDOT and GC/CNT electrodes data, and the third equivalent circuit is used for fitting the GC/PEDOT/CNT, GC/CNT-GRO and GC/PEDOT/CNT-GRO electrodes data.These circuits contain various elements: R s and R ct are the solution resistance, and the charge transfer resistance, respectively; R 2 and R 3 represent the different film layers resistances .Capacitance is represented by C. Also, two constant phase elements Y 1 ° and Y 3 ° are used to describe the capacitance, surface inhomogeneity and roughness of surface; n, m are their corresponding exponents (with values < one).Charge diffusion from bulk of solution to electrode surface is represented by Warburg impedance Y 2 ° (W).A quasi-semicircle part of the plot with large diameter at higher frequency region and a linear part at lower frequency region are depicted in Fig. 2 at bare GC corresponding to electron transfer-controlled and diffusion-controlled processes, respectively.The charge transfer resistance decreased for the working electrodes in the following order: bare GC > GC/PEDOT > GC/CNT > GC/CNT-GRO > GC/PEDOT/CNT > GC/PEDOT/CNT-GRO, manifesting relatively lower charge transfer resistance and fast charge transfer kinetics upon modification of GC electrode.GC has a higher charge transfer resistance 53.72 kΩ.Also, there is a significant decrease in the total impedance in the Nyquist plot of GC/PEDOT/CNT-GRO compared to bare GC depicting the effective performance and catalytic activity of the proposed sensor.Table 1 illustrates a summary of the EIS fitting data for the previously mentioned electrodes.Comparing the modified electrodes GC/CNT-GRO, GC/PEDOT/CNT and GC/PEDOT/CNT-GRO.A noticeable decrease in the value of Rct and R 2 are observed upon modification of the GC with PEDOT/CNT-GRO, confirming enhanced electron transfer process.Also, an increase in the value of Warburg W is observed upon modification reflecting fast diffusion kinetics.The components of the  www.nature.com/scientificreports/nanocomposite offer enhanced conductivity, and improved surface area of the proposed surface.An increase in the ionic accumulation at the surface of the proposed surface is confirmed by the high value of constant phase element Y 3 ° at GC/GC/PEDOT/CNT-GRO.

Electrochemistry of the working electrodes
The electrochemical behavior of HQ, CC, and NT/0.1 M PBS (pH 7.0) at modified GC electrodes, upon layerby-layer modification of GCE surface, is evaluated using cyclic voltammetry mode (CV).Figure 3 displays the CVs of a ternary mixture containing 200 µM HQ, 150 µM CC, and 100 µM NT prepared in 0.1 M PBS recorded at the following working electrodes: bare GC, GC/PEDOT, GC/PEDOT/GRO, GC/CNT-GRO, GC/PEDOT/ CNT and GC/PEDOT/CNT-GRO, with a scan rate of 50 mV/s.Supplement Fig. 6 shows the blanks for all the modified working electrodes.The electrochemical data for the oxidation of the studied analytes are summarized in Supplement Table 2.
Using the bare GCE or GC/PEDOT/GRO electrode, overlapped oxidation current signals for HQ and CC with low current response of NT are obtained for HQ, CC, and NT.Modifying the GC electrode surface with a thin film of PEDOT enhances the current signals and improves the resolution of the oxidation peaks compared to bare GC electrode.Then, modification of the GC electrode surface by two consecutive thin layers from PEDOT and CNTs, higher current responses are obtained for all the three analytes.The oxidation current responses are 40 μA, 31 μA, and 76 μA for HQ, CC, and NT at 83 mV 195 mV, and 791 mV, respectively.This improvement in current responses can be attributed to the synergistic effect between PEDOT and CNTs.The combination of these materials enhances the catalytic effect toward HQ, CC, and NT oxidation due to their good electric conductivity, and large surface area.Modification of the GC electrode surface by one layer of CNT-GRO mix, the oxidation current responses are 27 μA, 28 μA, and 32 μA for HQ, CC, and NT at 41 mV 145 mV, and 745 mV, Table 1.EIS fitting data corresponding to results in Fig. 2 GC electrode data' fitted with equivalent circuit 1. GC/PEDOT and GC/CNT electrodes data' fitted with equivalent circuit 2. GC/PEDOT/CNT, GC/CNT-GRO and GC/PEDOT/CNT-GRO electrodes data' fitted with equivalent circuit 3.  www.nature.com/scientificreports/respectively.But further modification of the GC electrode surface with two thin layers of PEDOT and CNT-GRO mixture enhances the sensor sensitivity and increases the oxidation current responses up to 106 μA, 82 μA, and 111 μA at 96 mV, 213 mV and 798 mV, respectively.An increase in the anodic current responses for HQ, CC, and NT is obtained with 5.1, 4.1, and 2.3 times higher compared to GC/PEDOT electrode, respectively.This is due to high electric conductivity of PEDOT and CNTs with a large active surface area of the PEDOT/CNT-GRO composite which resulted in acceleration of the electron transfer rate between the studied analytes and the composite surface.Thus, the synergistic effect between the modifiers GRO, CNTs, and PEDOT increases the electro-catalytic activity of the PEDOT/CNT-GRO composite toward the oxidation of HQ, CC, and NT.Besides, the conducting structure of the nanocomposite improves the voltametric separation between the HQ, and CC isomers that oxidize at nearly similar potentials under diffusion conditions and increases their oxidation current responses.The oxidation mechanisms of HQ, CC, and NT/0.1 M PBS (pH 7.0) using the GC/PEDOT/ CNT-GRO electrode are illustrated in Fig. 4 Effect of scan rate The effect of varying the scan rate (10 to 100 mV s −1 ) on the current responses of 100 µM HQ, 100 µM CC, and 120 µM NT/0.1 M PBS (pH 7.0) using a GC/PEDOT/GRO-CNT electrode is investigated via CV mode, Supplement Fig. 7.The inset A shows linear relationships between I p and ν 1/2 for HQ, CC, and NT, indicating diffusion-controlled processes.The corresponding equations for the linear relations of HQ, CC, and NT can be summarized as follows: Also, linear relationships between log I p and log v for HQ, CC, and NT are obtained, verifying that the processes are diffusion controlled, Inset B of Supplement Fig. 7.
The correlation slopes are 0.67, 0.71, 0.61 for HQ, CC, and NT respectively, close to the theoretical value of 0.5, indicating that the oxidation reactions are diffusion controlled processes 65 .
For the reversible electrochemical process, according to Laviron theory 66 , a graph of E p = f (log v) shows two linear lines with slopes equal to − 2.3RT/vnF for the cathodic peak and 2.3RT/(1 − v)nF for the anodic peak.The charge transfer coefficient (α) can be calculated based on the slopes of the two linear lines of E p versus log v relation using the following equation: where k a and k c are the slopes of the linear lines for E pa versus log v, and E pc versus log v, respectively.www.nature.com/scientificreports/ The relations between the anodic peak potential E pa and the cathodic peak potential E pc for HQ and CC versus the logarithm of the scan rate are depicted in inset C of Supplement Fig. 7 and can be represented by the following linear relationships: Thus, α values were calculated and found to be 0.55 and 0.65 for HQ and CC, respectively.Using the slope of E p versus log v, the calculated n values are equal to 2.21 and 2.36 for HQ and CC, respectively, indicating that two electrons are involved in the electrochemical oxidation of HQ and CC.
Also, a linear relationship between the anodic peak potential (E pa ) and the natural logarithm of the scan rate (log v) for NT is depicted in inset D of supplement Fig. 7 and is represented by the following linear regression equation.
For a totally irreversible electrode process, the relationship between E pa versus log v is expressed as follows by Laviron 67 : where R = 8.314 J/K mol, F = 96,480 C/mol, T = 298 K, and n is the total number of electrons exchanged.α is the electron transfer coefficient.α can be derived using the following formula 68 .
where E P/2 is the potential where the current is at half the peak value.α was calculated to be 0.793, The value of n can be determined as 1.95 = 2 using the slope of E p and log v.

Effect of pH
The performance of the electrochemical sensor is significantly affected by the pH of the electrolyte.To investigate the effect of pH on the simultaneous detection of HQ, CC, and NT in their mixture, DPV measurements are performed.The DPV responses of 200 µM HQ, 150 µM CC, and 100 µM NT are recorded in 0.1 M PBS within the pH range of 3 to 11, as shown in Fig. 5A.The results reveal that the oxidation peak currents of HQ, CC, and NT increase with increasing pH, and they reach their maximum value at pH 7.0 as shown in Fig. 5B.Therefore, pH 7.0 is chosen as the optimal condition for the subsequent experiments.In strongly acidic medium the nitrite is not stable, where it is easily undergoing a disproportionation reaction which leads to a decrease of nitrite oxidation current response 69 .The presence of hydrogen ions is necessary for the oxidation of nitrite 70 .Therefore, when the pH is higher than 7.0, the number of hydrogen ions decreased in basic medium, thus the nitrite oxidation is inhibited, and its oxidation peak current decreased, Fig. 5B.Several studies mentioned proposed mechanisms for nitrite oxidation.The mechanism suggests an electrochemical step (1) with the formation of NO 2 and followed by a chemical disproportionation reaction (2) and formation of NO 3 − (3) as follows 71 : The HQ, and CC compounds pKa values are 9.85, and 9.4, respectively 72 .Thus, at pH 7.0 there are good interactions between the partially negative charges oxygen atoms over the GRO surface (functional groups containing oxygen) and the protonated forms of HQ and CC compounds.At pH value lower or higher than 7.0, there are weak interactions between the oxygen atoms over GRO surface and the HQ and CC compounds.This is due to the presence of protons in acidic medium competing with the protonated compounds, and in basic medium the protonated compounds became neutral i.e., deprotonation of the protonated compounds occurred with the existence of OH -ions in the medium 18 .
Figure 5C illustrates that the anodic peak potentials (E p ) for HQ and CC shift negatively with increasing pH from 3 to 11, indicating the participation of protons in the electrochemical redox processes 73,74 .Additionally, the E p exhibits linear relationships with increasing pH for HQ and CC and can be represented by the following equations: www.nature.com/scientificreports/ The slopes of the regression equations are close to the Nernst theoretical value of 59 mV/pH, suggesting that the electrochemical redox reaction for each of HQ and CC at GC/PEDOT/CNT-GRO is two-protons and twoelectrons process 75 .Thus, the pH study provides a valuable insight into the electrochemical behaviors of HQ, CC, and NT, it can aid in the development of more accurate and reliable electrochemical sensors for their detections.

Reproducibility and stability
Reproducibility and stability are important parameters for precise and accurate analytical measurement.The relative standard deviation values (RSDs) of oxidation current responses for 100 µM HQ, 100 µM CC, and 120 µM www.nature.com/scientificreports/NT in their mixture using three similar fabricated GC/PEDOT/CNT-GRO electrodes are 1.23%, 1.73%, and 0.89%, respectively.Further, the stability of the GC/PEDOT/CNT-GRO sensor in a mixed solution of 100 µM HQ, 100 µM CC, and 120 µM NT/0.1 M PBS is evaluated.The RSDs of oxidation current responses obtained after 25 continuous CV cycles for HQ, CC, and NT in their mixture are 2.08%, 1.29%, and 2.83%, respectively, Supplement Fig. 8A.Also, the stability of the GC/PEDOT/CNT-GRO sensor in low concentration mixed solution of 2 µM HQ, 5 µM CC, and 3 µM NT/0.1 M PBS is examined.The RSDs of oxidation current responses obtained after 15 continuous DPVs for HQ, CC, and NT in their mixture are 2.038%, 1.84%, and 1.28%, respectively, Supplement Fig. 8B.Moreover, the GC/PEDOT/CNT-GRO electrode is stored for one month, the electrochemical current responses of HQ, CC, and NT maintain 95.3%, 98.2%, and 96.9% of their initial current values, respectively.These results suggest that the GC/PEDOT/CNT-GRO electrode has a good reproducibility, stability, and acceptable repeatability, making it a good choice for simultaneous detection of these pollutants species.

Robustness
The robustness of this method is assessed by examining the impact of minor changes in the experimental conditions.Two parameters are studied, namely the time before running the experiment (2 min ± 20 s) and pH change (7.0 ± 0.2).The RSDs for these parameters are found to be 1.96%, 1.56%, 1.42% and 2.0%, 1.96%, 1.82% for HQ, CC, and NT respectively, which confirm the steadiness of their current responses.These results suggest that the method is robust and can provide reliable and consistent results even with minor variations in the experimental conditions.

Simultaneous determination of HQ, CC, and NT in real water sample
The sensor's performance is further assessed using the DPV technique to determine HQ, CC, and NT simultaneously in wastewater sample under optimized conditions.The proposed method is validated according to ICH guidelines 76 .Pulse voltammetric methods, such as DPV, are effective and rapid electroanalytical techniques with well-established advantages, including good discrimination against background current and low detection limits.The following are the parameters for the DPV experiments: E i -200 mV, E f + 1000 mV, scan rate 20 mV/s, pulse width 50 ms, pulse period 200 ms, and pulse amplitude 10 mV.The HQ, CC, and NT are successfully detected simultaneously in wastewater sample with changing their concentrations as shown in Fig. 6A.Three distinct oxidation peaks appeared on the DPV, as the concentrations of HQ, CC, and NT increased in the ranges (0.04 → 100 µM), (0.01 → 100 µM) and (0.05 → 120 µM), respectively, their oxidation peak currents increased accordingly.Figure 6A; insets show linear fit curves between the oxidation peak current and the concentration for HQ, CC, and NT.The curves demonstrate that within the concentration range for each compound the peak currents increase linearly with their corresponding concentrations.The linear fitting equations between the oxidation peak current and the concentration for HQ, CC, and NT are as follows: The detection limits (DLs) are 8.5 nM, 3.8 nM and 6.1 nM, respectively.DLs are determined according to (S/N = 3).The GC/PEDOT/CNT-GRO sensor provides reasonable linear ranges for HQ, CC, and NT detection with DLs lower than other cited modified electrodes in previous work 17,69,[77][78][79][80] as illustrated in Supplement Table 3.The results indicate that the developed sensing method is appropriate for detecting HQ, CC, and NT simultaneously in wastewater sample without any cross-interference.

Investigating the intermolecular effect among HQ, CC, and NT
To investigate the intermolecular effect among HQ, CC, and NT, two experiments are conducted under optimized conditions.In the first experiment, the concentration of one analyte is changed while keeping the concentration of other analytes constant, and in the second experiment the reverse is made.Firstly, the concentration of NT is changed from 0.01 to 30 µM while the concentrations of HQ and CC are fixed at (20 µM) and (10 µM), respectively, Fig. 6B.Linear relationship is obtained between the peak current and the concentration of NT as shown in (Fig. 6B; inset) with the following regression equation: The DL for NT is 2.4 nM.In the second experiment, the concentrations of HQ and CC are changed from 0.06 to 40 µM and from 0.02 to 30 µM, respectively, while the concentration of NT is fixed at 60 µM, Fig. 6C.Linear relationships are obtained between the peak current and the concentration for CC and for HQ as shown in (Fig. 6C; insets) with the following regression equations:

Selectivity
The selectivity of the sensor plays a crucial role in its practical application.Therefore, the selectivity of the modified electrode is evaluated in the presence of potentially interfering species.The ability to detect HQ, CC, and NT in real water samples in presence of various cations and anions such as K + , Mg 2+ , Cu 2+ , Na + , Cd 2+ , Pb 2+ , Mn 2+ , Ca 2+ , SO 4 , Cl − , and PO 4 3− with 100-fold concentration has been investigated using the GC/PEDOT/CNT-GRO electrode.No significant variation of the anodic peak current (less than 4.55%) and the oxidation potential values of HQ, CC, and NT in presence of these interfering ions.Also, the current responses of the sensor show less than 5.56% variation in presence of 50-fold concentration of interfering compounds such as 3-aminophenol, aniline, phenol, and citric acid, Fig. 7.These results demonstrate the anti-interference capability of the proposed surface and a good selectivity for simultaneous detection of these pollutant species.

Recovery
We studied the practical feasibility of the proposed method for determination of HQ, CC, and NT in wastewater sample by standard additions using the GC/PEDOT/CNT-GRO sensor.The DPV current responses of HQ, CC, and NT in wastewater sample are measured after each addition of known concentrations of HQ, CC, and NT.Each run is repeated three times after each addition and the average current responses for HQ, CC and NT are calculated.The results of the calculated concentrations and the recoveries are presented in Table 2.The recovery for HQ, CC and NT ranged from 97.7 to 101.2%, 98.9 to 102.4% and 98.1 to 102.3% respectively, indicating that the GC/PEDOT/CNT-GRO sensor has excellent recovery rate for HQ, CC, and NT determination in wastewater sample.These findings demonstrate that the GC/PEDOT/CNT-GRO electrode is an effective, reliable, and accurate for detecting these pollutants in wastewater samples.

Conclusions
A novel method is developed for simultaneous detection of three environmental pollutants, namely HQ, CC, and NT using GC/PEDOT/CNT-GRO electrochemical sensor in real water samples.The modification of the GC electrode with PEDOT/CNT-GRO composite resulted in improvement of the electronic transport rate between the studied pollutant species and the modified composite surface.The sensor detects the HQ, CC, and NT in wide concentration ranges (0.04 → 100 µM), (0.01 → 100 µM) and (0.05 → 120 µM) with low detection limits of 8.5 nM, 3.8 nM and 6.1 nM, respectively compared to other working electrodes reported in the literature.The synergistic effect between the modifiers GRO, CNTs, and PEDOT increases the electro-catalytic activity of the PEDOT/CNT-GRO composite toward the oxidation of HQ, CC, and NT.Besides, the advantageous structural, morphological, conducting, and other specific properties of the nanocomposite improve the voltametric separation between the HQ, and CC isomers that oxidize at nearly similar potentials at conventional electrodes.Good potential peak separations: 117 mV and 585 mV are obtained between the HQ-CC, and CC-NT.Further, the sensor has perfect stability, and reproducibility, good anti-interference capability and excellent selectivity for the simultaneous determination of HQ, CC, and NT in presence of common interfering ions and compounds in wastewater samples, making it a reliable tool for detecting these pollutant species in complex environmental samples.Thus, the sensor has a good impact for actual practical applications.

Figure 4 .
Figure 4. Schematic representation of GC/PEDOT/CNT-GRO modified electrode used for the electrochemical oxidation of HQ, CC, and NT.

Figure 5 .
Figure 5. (A) DPVs of ternary mixture of 200 µM HQ, 150 µM CC, and 100 µM NT/0.1 M PBS with different pH values, scan rate 20 mV/s using GC/PEDOT/CNT-GRO electrode.(B) Relations between oxidation peak currents and pH for CC, HQ, and NT.(C) Relations between oxidation peak potentials and pH for CC and HQ.

Figure 6 .
Figure 6.(A) DPVs for simultaneous determination of HQ, CC and NT in concentration ranges (0.04 → 100 µM), (0.01 → 100 µM) and (0.05 → 120 µM) in wastewater/0.1 M PBS pH 7.0; insets: the corresponding calibration curves for HQ, CC and NT using GC/PEDOT/CNT-GRO electrode.(B) DPVs of NT in the concentration range from (0.01 μM to 30 μM) in presence of constant concentration 20 μM of HQ and 10 µM of CC in wastewater/0.1 M PBS pH 7.0; inset: the corresponding calibration curve for NT.(C) DPVs of HQ and CC in the concentration ranges from (0.06 μM to 40 μM) and (0.02 μM to 30 μM) in presence of constant concentration 60 μM of NT in wastewater/0.1 M PBS pH 7.0; insets: the corresponding calibration curves for HQ and CC.

Table 2 .
Evaluation of the accuracy and precision of the proposed method for determination of HQ, CC, and NT in wastewater sample.